Broadly, the present invention relates to mutating starch genes in polyploid cereal grains. Specifically, this invention concerns mutant wheat plants, mutant wheat grain and the starch therefrom.
Cereal grains, such as rice, wheat, corn and barley help to feed the world""s population. A large percentage of the world population""s staples are formed of these grains. These grains are processed to make breads, cereals, pasta, flour, etc. Processed grains have different qualities which lead to different product uses. Wheat grains are processed into wheat flour, which is a storehouse of nutrients. The starch, protein, lipids, enzymes, and nutrients affect, in differing degrees, the flour product. Starch in the flour product affects its characteristics to a large degree. The digestibility, processing temperatures, cooking qualities of flour are all impacted by the type of starch used.
Starch is formed of two components amylose and amylopectin. At least one of these components is altered in a number of diploid cereal grains which have starch mutations. One starch mutation is referred to as the waxy mutation. Cereal grains having the waxy mutation form low amylose starch. Naturally occurring waxy mutants are well known in rice and maize, both diploid species. However, naturally occurring starch mutants are not known in polyploid species. To alter the starch in a polyploid species requires several independent mutations. Wheat seldom has naturally occurring mutants because both soft and hard wheats are hexaploid, although durum wheats are tetraploid. No one has discovered a naturally occurring waxy wheat.
Wheat (Triticum aestivum L.) has three chromosome sets derived from three different species. Each chromosome set has a genome letter, A, B or D. A wheat waxy mutant would have homozygous waxy alleles in each of the A, B and D chromosomes. Mutations in wheat have been identified by protein characterization in the individual A, B and D genomes. Until 1992 these protein characterizations detected only a single waxy protein band. The test was not able to distinguish a protein band for each of the three genomes. If one of the three genomes was not producing a protein this test could not detect it. A modified detection system using SDS-PAGE with low BIS acrylamide concentration and a two-dimensional gel electrophoresis (2-D PAGE) showed two of the expected three protein bands. The third band was detected by using isoelectric focusing (IEF) for the first dimension and the modified SDS-PAGE for the second. The modified detection system detected three protein bands. Each protein band corresponds to one of the A, B and D sets of chromosomes. By detecting the individual proteins wheat lines could be screened for null waxy alleles. A null allele does not produce a certain protein at that allele on a certain chromosome. A null mutant does not produce a certain protein at any of the chromosomes. This is in contrast to a non-null mutant which does produce the protein, but in an inactive state.
Once a test showing the three bands was identified a number of researchers began to screen waxy wheat for null alleles. Single null alleles were located when individual starch proteins were missing from wheat starch. Single null waxy wheat alleles were identified in only about 10% of the US winter wheat germplasm. The remaining wheat were wildtype having three functional wx loci. R. A. Graybosch reported on a few single null waxy alleles in both the A and B genomes. Only two single nulls are known to exist in the D genome. One single null D genome waxy allele has been reported in Japan and one has been reported in Canada.
Recently, researchers discovered four separate double null waxy alleles in wheat. Each of these double null waxy alleles (partial mutants) were null for waxy alleles of the A and B genome. The Japanese reported the Kanto lines 79, 107, Saikai 173 and R. A. Graybosch reported Ike. Ike is a public line, developed by the Kansas University breeding program. These double null waxy partial mutants are the only ones known to exist. However, with the modified screening procedure researchers can expect the discovery of additional single and double null alleles. Like single waxy nulls, these newly discovered double null waxy partial mutants, still produce a waxy protein in the D allele and thus still have significant amylose content. However, the double null waxy partial mutants amylose content is recognizably less then single waxy nulls.
Even after the discovery of the double nulls there still was needed a waxy wheat mutant plant that had a waxy mutation in all three genomes. In 1994, a waxy wheat plant free of waxy protein was produced. The Bai Huo Chinese cultivar lacking a waxy protein was crossed on to Kanto 107 and Saikia 173. Out of 720 F2 seeds 14 were free of Wx proteins.
Conventional crossing of a partial null mutant with a single null allele did produce a waxy wheat. It also produced an entirely new combination of genes within the chromosomes of the resultant waxy wheat. To generate useful lines from the resultant waxy wheat plant breeding was used. The waxy wheat was selfed and the progeny were selected for agronomics traits and the waxy trait throughout breeding generations. Although conventionally bred plants having the waxy mutation are easily identified by the iodine test; agronomic traits are much more difficult to identify. Agronomic traits are often multigenetic and in wheat these are further complicated by three separate sets of chromosomes. Reconstruction of the three chromosomes with conventional breeding, or even with dihaploidy takes time and a number of crosses. Even after a number of crosses, the waxy wheat is not essentially identical to one parent, it is only similar to the parent.
A plant which is essentially identical to the parent plant is an isogenic line. An isogenic line is characterized by essentially identical genes. Forming a waxy wheat that is isogenic to its parent avoids conventional breeding problems. There is a need for a method of forming a waxy wheat that does not produce an entirely new combination of genes within the chromosomes of the waxy wheat. There is a need for an efficient method for forming full mutants from double chromosome mutants or double chromosome mutants, from single chromosome mutants in polyploid cereals. In other words, there is a need for a method to form isogenic polyploid lines which contain mutations.
Additional needs develop, once an isogenic starch mutation is in commerce. Breeders will breed with this isogenic germplasm to place the starch mutation into other germplasm. To maintain germplasm security, there is a need for a method of identifying the mutation. If the mutation is identified then misuse of germplasms can be identified.
An object of the present invention is a method of forming polyploid isogenic seeds and plants which differs from the parent by being a starch mutant.
Still another object of the present invention is an isogenic wheat seed and plant that produces waxy starch.
A further object of the present invention is a method of identifying isogenic lines by fingerprinting the modified starch the lines produce.
Yet a further object of the present invention is waxy wheat starch which contains an inactive protein in at least one of the waxy loci.
In addition, another object of the present invention is the method of mutating polyploid cereals to form a modified starch.
The present invention broadly encompasses a method of making a full mutant allele polyploidy cereal seed. This method includes the following steps. Treating a double mutant allele in a polyploid plant material with a mutagen. This forms treated plant material. The treated plant material is then screened to identify full mutant allele plant material. The full mutant allele plant material are selected. The present method can also include the additional steps of selecting polyploid plant material having a single mutant allele and treating this plant material with a mutagen then screening the plant material to identify plant material that contain a double mutant allele. This plant material is then used in the earlier method to form the full mutant allele. A number of mutagens can be employed but the EMS mutagen is preferred as it makes point mutations. The method can be performed on plant material such as wheat seeds. In this method the double mutant allele can be a double null allele or the double null allele can be a double null waxy allele. The screening can include a test for opaqueness. The step for screening can also include testing starch with iodine. The full mutant allele seeds stain red when tested with iodine.
The present invention also encompasses a product. A polyploidy plant material having either no proteins or inactive protein produced in one allele in all sets of chromosomes and at least one of the alleles containing a point mutation due to application of mutagens. This plant material can also include at least one allele in one set of chromosomes producing an inactive protein due to said point mutation.
The polyploid plant material in accordance to the present invention includes isogenic polyploid plant material that contains at least one mutagen induced point mutation in a specific allele in at least one set of chromosomes and at least one naturally occurring mutation in the same specific allele in a different set of chromosomes. In addition the present invention includes the starch produced there from. The progeny of this plant material is within the scope of the present invention. More specifically, the present invention encompasses double null mutant alleles in polyploidy plant material that is formed into full mutant alleles by mutagens. The present invention particularly includes a waxy 60 mutant plant material and the starch therefrom. This waxy 60 mutant can be formed from a number of waxy partial mutants. Specifically it can be formed from IKE. The present invention includes isogenic lines to a wheat parent line that contains a fill mutant waxy allele. Particularly, the present invention encompasses isogenic lines to IKE, Kanto lines 79, 107, Saikai 173 and Bia Huo that contain a full mutant waxy allele and the waxy starch therefrom. The present invention also includes a isogenic wheat plant with a full mutant starch allele and at least one fingerprint protein.
The present invention also includes a method of identifying ancestry of polyploid material by isolation of the fingerprint proteins of the starch. Additionally, the present invention includes the method of identifying the fingerprint protein in the starch of waxy wheat.
Single null *allelexe2x80x94no protein produced in one allele in one set of chromosomes.
Double null *allelexe2x80x94no protein produced in one allele in two sets of chromosomes.
Full null *allelexe2x80x94no protein produced in one allele in all sets of chromosomes.
Single inactive *allelexe2x80x94an inactive protein produced in one allele in one set of chromosomes.
Double inactive *allelexe2x80x94an inactive protein produced in one allele in two sets of chromosomes.
Full inactive *allelexe2x80x94an inactive protein produced in one allele in all sets of chromosomes.
Single mutant *allelexe2x80x94no protein produced in one allele in one sets of chromosomes or an inactive protein produced in one allele in one set of chromosomes.
Double mutant *allelexe2x80x94no protein produced in one allele in two sets of chromosomes or an inactive protein produced in one allele in two sets of chromosomes.
Full mutant *allelexe2x80x94no protein produced in one allele in all sets of chromosomes or an inactive protein produced in one allele in all sets of chromosomes.
* Identifying terms may be added such as starch, waxy, ae, dull, sugary2, etc. 
Waxy partial mutantxe2x80x94having no waxy proteins or inactive waxy proteins produced in one allele in two sets of chromosomes.
Waxy 60 mutantxe2x80x94having either no waxy proteins or inactive waxy protein produced in one allele in all sets of chromosomes but at least one allele in one set of chromosome producing an inactive waxy protein.
New full mutantxe2x80x94having either no proteins or inactive protein produced in one allele in all sets of chromosomes but at least one allele in one set of chromosome producing an inactive protein.
Broadly, the present invention produces mutated starch plants by mutagenesis, in polyploid cereal grains. Such new starch mutant plants are produced more efficiently than starch mutant plants produced by conventional breeding or even biotechnology-aided breeding. The mutant plants of the present invention alter the starch. One type of altered starch is waxy starch. This invention particularly includes the production of waxy polyploid plants. More specifically this invention includes waxy wheat plants. More specifically waxy 60 mutant polyploidy grain.
The waxy wheat plant invention is formed from a double null waxy allele wheat mutated with ethyl methane sulfonate (EMS). The resultant waxy 60 mutant wheat plant produces waxy starch containing a 60 kDa bound starch synthase protein (it is not null for all 3 waxy alleles). This inactive 60 kDa bound starch synthase protein identifies the present invention. The present invention can be distinguished by the presence in starch extracts of this inactive 60 kDa bound starch synthase protein from existing waxy wheat. Like all waxy wheat starch, the wheat starch of the present invention stains red with iodine. This red stain identifies the starch as waxy starch. Unlike all presently existing waxy wheat starch, the present invention has a 60 KDa waxy protein band that appears when the starch proteins are extracted and separated by SDS-gel electrophoresis. This fingerprint protein of the present invention is also identifiable by use of antibodies specific to the waxy protein.
In rare instances, the EMS mutation may stop the 60 KDa protein from forming in the starch. This rare plant would be a triple null in the waxy allele without a fingerprint. A triple null waxy plant and a plant with a double null with an inactive 60 KDa protein in the waxy allele produce the same waxy starch with the exception of the presence of the 60 KDa protein in the starch of the later. Although in this rare instance the waxy starch would appear the same as the prior art starch the plants would still be very different. Isogenic plants are plants that have essentially identical genes. Plants of the present invention are isogenic and plants formed by breeding and haploidy are a mixture of genes from two sources. The term plant includes all plant parts including cells, leaves, roots, meristems, stems, flowers, seeds, and pollen.
The isogenic line of the present invention once produced can be bred through traditional breeding methods or marker breeding methods to move the starch mutant alleles into different wheat germplasm, or to move different alleles into the starch mutant wheat. The waxy trait is retained in the breeding process if the iodine stains red.
The present invention is a repeatable method of mutating polyploid plants to form isogenic plants with starch mutations. A polyploidy plant having at least one starch mutation in one set of chromosomes is selected. This plant is mutated by EMS. This method of mutagenesis forms an isogenic plant with a point mutation. The resultant plants are screened for the desired point mutation that give the desired starch. If the waxy mutation is the desired mutation then the screening is for opaqueness of the seed. Further screening of these seeds can be achieved by staining the starch in the seed with iodine. Seeds with starch that stains red are waxy.
Generally then the method of the present invention is selecting a polyploid plant with an existing starch mutation in at least one set of chromosomes, mutating the selected plant and screening for the mutation in the other set of chromosomes. Each step of the method can vary slightly. For example the selection can be done by a protein isolation test such as the modified SDS-PAGE, or by a phenotype test. Once selected, the germplasm may have to be increased to have sufficient plant material for the mutation step.
The mutation step can be done by a number of mutagenesis methods. These methods mutate plant material with a suitable mutagens. These mutagens can be used on the pollen, the fruit, the anthers, the seeds and the ovum of different plants. The most preferred method is treatment of seed or pollen with the mutagens. The mutagens can be, chemical or physical. Chemical agents include but are not limited to ethyl methane sulfonate, diazo reagents, N-nitroso, N-methyl glycine, psoralens and physically by ultraviolet light, X-rays, gamma ray and any other agents having similar effects may also be used. The more preferred group of mutagens includes the use of sodium azides or nitrosoguanidine, or any alkylating agent like ethyl methane sulfonate (EMS). The most preferred agent being EMS. The EMS method is outlined in the Neuffer paper in the Maize Genetic Newsletter 45 PAGE 146 (1971).
The EMS method generates a point mutation in the nucleotide sequence of the gene. Some of the other methods cause more gene disruption than a point mutation. EMS mutates more specifically and does not change most of the plant""s genome. The present invention thus forms lines that are isogenic to the parent line and include the new point mutation. The point mutation is an inheritable genetic change in the DNA of the plant. The genetic change is called an induced mutant allele which means a mutation in the plant genome that was introduced into the plant or an ancestor of the plant by application of a mutagens and not by transformation.
The final step is screening to identify plants grown from the mutated seeds that carry the starch mutation in the additional set of chromosomes. The screening can be based on phenotypic traits, starch components, starch characteristics and the like.
This general method of forming isogenic lines from polyploid cereals was used to form a waxy wheat isogenic to IKE. In this specific use of the method, IKE, a double null waxy allele, (an A,B double null wheat seed) was selected. Ike seed was planted and the self pollinated seed was harvested. The harvested grain (or seeds) were mutated with EMS. The number of treated seeds was large enough to produce the desired mutation events. The probable number of desired mutation events is a function of genetics and polyploidy. This number can be calculated by one of ordinary skill in the art. When Ike was mutated, the desired event, which is a point mutation in the waxy allele of the D genome occurred, in one out of every 600 plants. These plants carried a single inactive waxy allele and a double null waxy allele. To maximize the waxy 60 mutant plants (having an additional mutation in the waxy allele of the D genome), 15 pounds of Ike seeds were mutated in an EMS solution for 16 hours then rinsed thoroughly. The treated seeds were planted. The wheat seed was grown, fertilized, herbicide-treated, and insecticide treated as was appropriate. The wheat when ready to harvest was hand-harvested. Mechanical harvesting can be used, but may result in less useable seeds. The hulls on the seeds were removed for seed screening. The seeds were screened for the desired starch trait by viewing the seed on a light box and approximately 60 opaque seeds were selected from a small number of seeds. These selected seeds were then stained with iodine. Distant from the embryo, the seeds were cut with a razor. The iodine was applied to the cut portion of the seed. If the stain was red the seed was waxy wheat, if the stain was not red, the seed was discarded. The selected waxy 60 mutant IKE seeds were planted to increase seed.